A detailed model ofthe molecular mechanisms by which small, enveloped viruses infect humans and other mammals and insects is a long-range goal of our collaborative PPG studies. In Project 1, the methods of electron cryo-microscopy (cryoEM), three-dimensional (3D) image reconstruction, and pseudo-atomic modeling will be used to explore the structures of Sindbis virus, and other alphaviruses such as Ross River and Aura virus. None of these have been crystallized in a form suitable for X-ray diffraction studies. The El membrane-anchored glycoproteins (Class II fusion proteins) of alphaviruses promote fusion of viral and host endosomal membranes in a manner quite distinct from the mechanism carried out by Class I fusion proteins typified in the influenza and HIV systems. Determination of alphavirus structures at sub- nanometer resolutions will enable detailed pseudo-atomic models to be constructed and help determine how the viral glycoproteins are organized, how they participate in low pH triggered fusion, and how, through interactions with the nucleocapsid core, they help direct assembly and budding of native virions. Dedicated efforts to improve the methods of specimen preparation, imaging, processing, and visualization are required to markedly improve the detail revealed in cryoEM structure determinations and will potentially enable a variety of related structures, such as site-directed mutants with defined phenotypes, to be studied similarly. We currently have achieved 7.2-A resolution in our studies of Sindbis, in part because we have developed an automated system by which 3D reconstructions at sub-nanometer resolutions can be obtained in times ranging from hours to a few days once image data are recorded. This new capability makes it feasible to study a large number of different virus samples and also to continue improving the methods to routinely achieve higher resolutions and hence build more accurate models. Work directed at performing 3D reconstructions directly at the electron microscope at low-resolution will further enable rapid identification of the most promising samples. RELEVANCE (See instructions): Alphaviruses are a group of ~26 arthropod-transmitted viruses, some of which produce asymptomatic infection in humans. Others produce fever, rash, and encephalitis in humans and domesticated animals, some with high mortality rates. They have been intensively studied both because of their utility as gene expression vectors for mammalian and insect cell lines and their major impact on human and animal health.